For the ionization of analyte ions by matrix-assisted laser desorption, the samples consisting of matrix substance with small numbers of embedded analyte molecules, are bombarded with short pulses of light from a UV laser. Each pulse of laser light generates a plasma cloud of desorbed sample material. When the pulses of laser light are of moderate power, practically only molecular ions are created from the analyte molecules in the plasma cloud, not fragment ions, and therefore several types of analyte substance can be present in the sample and recognized simultaneously by their mass—in other words, mixture analyses can be carried out.
The method of ionization by matrix-assisted laser desorption is, in general, used to investigate large biomolecules, particularly large biopolymers such as, primarily, proteins or peptides obtained from proteins by enzymatic digestion, which yield mass spectra that can be evaluated effectively above 1000 Daltons. It is also possible to investigate their conjugates with sugars (glycopeptides) or fats (lipopeptides) in this way. (The spectrum in the range below 1000 Dalton usually is covered by very strong background noise and cannot be evaluated).
The only information contained in the mass spectra of the molecular ions is the molecular weight of the analyte molecules; there is no information about their identity or internal structure. Although it is possible to identify the proteins from the molecular weights of their digestion peptides by comparison with virtually digested proteins in a protein database, this identification does not offer a high degree of certainty. Modifications of the proteins can only be very approximately found by this method. Protein sequences, and also the structures of the conjugates, can only be recognized by recording the mass spectra of daughter ions obtained by fragmentation of the analyte ions in question.
In MALDI time-of-flight mass spectrometers, two different kinds of fragmentation processes can be carried out in order to generate daughter ions and, particularly in the case of proteins and peptides, they lead to different fragmentation patterns. The two types of fragmentation are referred to as ISD (“in-source decay”) and PSD (“post-source decomposition”). ISD requires the analysis of pure substances in relative high amounts, it is applied rarely up to now. Therefore, the acquisition of daughter ions generated by PSD is the predominant method.
To acquire daughter ion spectra created by PSD, the intensity of the laser light (energy density or fluence) is increased. As a result, a large number of unstable analyte ions are generated which, after their acceleration in the ion source, decompose with characteristic half-lives during their flight through the mass spectrometer, so forming daughter ions (also known as fragment ions). The unstable ions that decompose in the flight path of the mass spectrometer are often referred to as “metastable” ions. Recording the PSD daughter ion spectra, which in the past was a very complicated process that was only done piece by piece throughout the full mass spectrum, is nowadays carried out in one go in time-of-flight mass spectrometers specially designed for this purpose. Such a mass spectrometer is described in U.S. Pat. No. 6,300,627 B1.
FIG. 1 schematically illustrates a MALDI time-of-flight mass spectrometer for acquiring daughter ion spectra. A pulsed UV laser 3 sends a pulse of laser light through a focusing lens 4 and a deflecting mirror 5 onto the sample 6, which is located on a sample support 1 in a dried state. A small amount of the sample material abruptly evaporates, forming a plasma cloud. The ions in the plasma cloud include a great excess of matrix complex ions of every mass up to around 1000 Daltons, generating in the mass spectrum a huge background noise. The embedded analyte ions can therefore be measured effectively only in the higher mass range from about 1000 Daltons to 5000 Daltons. Accelerating potentials at the acceleration diaphragms 7 and 8 form the ions into an ion beam 9. The application of moderate accelerating voltages give a relatively low kinetic energy of only, for instance, 6 keV to the ions. The ions, therefore, are relatively slow and need some time up to the parent ion selector 10. An accelerating voltage that is switched on with a delay relative to the flash of laser light provides time-focusing of ions of the same mass at the location of the parent ion selector 10. This parent ion selector 10 is a bipolar switchable grid that only allows ions through in a straight line during an adjustable switching time window, so making them available for further analytical investigation. The parent ion selector is thus used to select the parent ions whose daughter ions are to be measured. If metastable parent ions have already decomposed between the acceleration diaphragm 8 and the parent ion selector 10, the daughter ions created here can also pass through the parent ion selector, because they have the same velocity as the undecomposed parent ions, and therefore arrive at the parent ion selector at the same time as they do.
The undecomposed parent ions and the daughter ions that have been created through the decomposition of parent ions now fly on to a post-acceleration unit 12, where they are given an additional acceleration by about 20 kilovolts. Prior to the post-acceleration, the daughter ions only possess a fraction of the energy of the parent ions, corresponding to their mass fraction relative to the parent ion. The post-acceleration now gives all the ions additional energy, causing their total kinetic energy to rise to between 20 and 26 kiloelectronvolts, which is particularly favorable for their further flight through the time-of-flight mass spectrometer for mass analysis. The mass analysis, in turn, is carried out by analyzing the time of flight at the detector 17, since the lighter ions, even if somewhat lower in energy, are faster and also reach the detector more quickly along the shorter beam 15 than the more energetic, but slower, ions traveling along the beam 16 that enters more deeply into the reflector 14.
There are different ways of achieving the post-acceleration. As a first approach, the selected ions can be made to fly through a small housing 12, whose potential is raised by about 20 kV while the ions pass through it, so that they are given their acceleration as they leave this housing. As a second approach, however, the entire flight path up to the post-acceleration point can be held at a high base potential of 20 kV. The initial acceleration of 6 kV must therefore be raised above this base potential. In this case, the high post-acceleration voltage of 20 kilovolt does not need to be switched. On the other hand, the initial flight path, including the parent ion selector and a collision cell (if present), must be held continuously at a high potential. This is achieved by locating the flight path of the ions in a long housing that is sealed on all sides, for instance in a tube 20 that is at this potential. The potential of this long tube is kept constant over time, and is not switched.
Daughter ions generated by decompositions of the already post-accelerated parent ions disturb the daughter ion spectrum and must be prevented from being recorded. To prevent these daughter ions from reaching the reflector 14, a further ion selector 13 is included in the ion path between the post-acceleration unit 12 or tube 20 and the reflector 14. This further ion selector 13, called “parent ion suppressor” suppresses the parent ions and their equally fast late daughter ions, as is described in U.S. Pat. No. 6,717,131. This parent ion suppressor 13 is not only necessary to suppress the daughter ions already created after the post-acceleration, but also to suppress the continuous background that would be generated by the daughter ions from parent ions that decompose at a random potential in the reflector 14.
Increasing the laser fluence is only one way to create daughter ions. Alternatively, the daughter ions can be generated by impacts with gas molecules in a collision chamber positioned somewhere between the first acceleration of the ions by the diaphragm 8 and the post-acceleration unit 12. The collision chamber is filled with collision gas at a suitable pressure, and generates fragment ions through the absorption of energy by a number of collisions (CID=collisionally induced decomposition).
In both types of these PSD-MALDI mass spectrometers for recording daughter ion spectra according FIGS. 1 and 2 it is therefore necessary to select the parent ions whose daughter ion spectra are to be acquired. Only relatively few daughter ions are created by each pulse of laser light, so that usually a suitably large number of individual daughter ion spectra is acquired with a few hundred up to a few thousand pulses of laser light, and to sum up these spectra, after the ion signals have been amplified and digitized, to form a sum spectrum of the daughter ions. The daughter ion sum spectrum then covers a sufficiently wide range of intensities to measure different species of ion with large differences in concentration.
When the term “daughter ion spectra” is used below, it follows that an individual daughter ion spectrum is meant when a procedure is being described that results from a single pulse of laser light, and that a daughter ion sum spectrum is meant when referring to the acquisition of spectra in general, for which necessarily a large number of laser light pulses are used.
In the analysis of mixtures, such as the analysis of the 20 to 30 digestion peptides resulting from a large, enzymatically digested protein, it is often desirable to record a daughter ion spectrum for each analyte substance, which in this case means for each digestion peptide. If something like 1000 pulses of laser light are required for each daughter ion spectrum, this means that the sample must be sufficiently large to generate between 20,000 and 30,000 desorption plasma clouds, each created by a strong pulse of laser light. This is often not the case when sample quantities are small.
In addition, the acquisition of such a large number of daughter ion spectra requires a great deal of time. A measurement time of several days is often required to analyze entire proteomes with reliable identification of all the proteins and their post-translational modifications by means of daughter ion spectra.
The term “mass” here always refers to the “charge-related mass” or “mass-to-charge ratio” m/z, which alone is relevant for mass spectrometry, and not simply the “physical mass”, m. The dimensionless number z represents the number of elementary charges, that is the number of excess electrons or protons on the ion that have an external effect as an ionic charge. Without exception, all mass spectrometers can only measure the mass-to-charge ratio m/z, not the physical mass m itself. The mass-to-charge ratio is the mass fraction per elementary charge on the ion. Correspondingly, “light” or “heavy” ions always refer to ions with a low or high mass-to-charge ratio m/z. The term “mass spectrum” again always refers to the mass-to-charge ratios m/z.